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Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase.

ABSTRACT: In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNA(Gln) is primarily synthesized by first forming Glu-tRNA(Gln), followed by conversion to Gln-tRNA(Gln) by a tRNA-dependent amidotransferase. We report a functional and structural analysis of the NTD of Saccharomyces cerevisiae GlnRS, Gln4. Yeast mutants lacking the NTD exhibit growth defects, and Gln4 lacking the NTD has reduced complementarity for tRNA(Gln) and glutamine. The 187-amino acid Gln4 NTD, crystallized and solved at 2.3?Å resolution, consists of two subdomains, each exhibiting an extraordinary structural resemblance to adjacent tRNA specificity-determining domains in the GatB subunit of the GatCAB amidotransferase, which forms Gln-tRNA(Gln). These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNA(Gln), consistent with a hinge-closing mechanism proposed for GatB recognition of tRNA. Our results suggest a possible origin and function of the NTD that would link the phylogenetically diverse mechanisms of Gln-tRNA(Gln) synthesis.

SUBMITTER: Grant TD

PROVIDER: S-EPMC3333875 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase.

Grant Thomas D TD Snell Edward H EH Luft Joseph R JR Quartley Erin E Corretore Stephanie S Wolfley Jennifer R JR Snell M Elizabeth ME Hadd Andrew A Perona John J JJ Phizicky Eric M EM Grayhack Elizabeth J EJ

Nucleic acids research 20111217 8

In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNA(Gln) is primarily synthesized by first forming Glu-tRNA(Gln), followed by conversion to Gln-tRNA(Gln) by a ...[more]

PMID: 22180531

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Project description:Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl-tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the KM and kcat obs , governing interactions with tRNA, were 1.0 μM and 0.15 s-1 , respectively. The crystal structure of the α2 form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three-dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad-spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild-type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time-kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 μg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.

Dataset Information

Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase.

Publications

Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase.

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